This research aims to exploit recent advances in ultrasensitive fluorescence microscopy to study protein trafficking into the nucleus mediated by "classical" nuclear localization signals (NLS). We propose to apply cutting edge non-invasive bioimaging technologies to quantify the real-time dynamics, mobility, and interactions of NLS cargoes and nuclear import receptors both in vitro and in their native live cell environment. The broad, long term objective of this research is to enhance our current understanding of the biophysical mechanisms involved in NLS dependent import of proteins into the nucleus. The specific aims include 1) Directly quantifying the protein-protein interactions between NLS cargoes and import receptor proteins in vitro; 2) Quantifying the assembly of NLS cargo / import receptor complexes in living cells; 3) Quantify the mobility and localization of NLS cargoes, and the spatial dependence of their interactions and mobility in living cells; and 4) Measure the conformational dynamics of the auto-inhibitory domain of importin-alpha nuclear receptor protein. The specific research aims outlined in this proposal will produce important health related results by leading to a deeper understanding of the nuclear import process. Regulation of nuclear import is clearly an important control point in signal transduction and malfunctions in these mechanisms are important in various human diseases. For example, a recent study identified a truncated form of importin-alpha nuclear import receptor expressed in the human breast cancer cell line ZR-75-1. While the biochemistry of the nuclear import process has been investigated in great detail, very little is known about the actual mechanism by which proteins are targeted to the nucleus. These studies, which will quantify the protein-protein interactions, intracellular distribution, mobility, and conformation dynamics of NLS cargoes and import receptor proteins in vivo are likely to lead to significant new insight into the biophysical mechanisms involved in this critical cellular function.